Chemical modification of the active site sulfhydryl group of saccharopine dehydrogenase (L-lysine-forming).

Saccharopine dehydrogenase (e-N-(L-glutaryl-2)-L1ysine:NAD oxidoreductase (L-lysine-forming) EC 1.5.1.7) is inhibited by a variety of sulfhydryl reagents. Althoughp-chloromercuribenzoate and 5,5’-dithiobis(2nitrobenzoate) react with three sulfhydryl groups present in the enzyme, reaction with one reactive sulfhydryl group leads to complete loss of catalytic activity. Amino acid analysis of the enzyme carboxymethylated by iodoacetate shows the formation of 1 eq of S-carboxymethylcysteine. The loss of one sulfhydryl group titratable with 5,5’-dithiobis(2-nitrobenzoate) after carboxymethylation confirms the site of modification as a cysteine residue. The inhibition by iodoacetate follows second order kinetics, with a rate constant of 0.2 mM-’ min-’ at pH 8.5 and 24°C. Modification of 1 cysteine residue results in total inactivation. The inhibition by iodoacetate is protected by the coenzyme (or analogs) and the coenzyme plus substrate (or analog). The effectiveness of protection by these compounds closely parallels their affinities for the enzyme, indicating that the modification occurs at the active site residue. The modification of the cysteine residue appears to result in a loss of ability to bind the coenzyme. Among alkylating agents tested, N-ethylmaleimide and iodoacetamide are stronger inhibitors than iodoacetate, as evidenced by their second order rate constants for inhibition.